Epidermal growth factor (EGF) has been shown to stimulate a variety of biological activities in osteoblastic cells, but the signaling pathways by which this mitogen exerts its activities has not been clearly defined. The activation of phospholipase C (PLC) leading to increases in inositol trisphosphate (IP3) by agents such as thrombin is a well know signaling pathway in osteoblastic cells. The activation of phospholipase D (PLD) by receptor-mediated hydrolysis of phosphatidylcholine is a major signaling pathway of EGF in a variety of cell systems. The purpose of the present study was to determine whether EGF stimulated PLD activity in normal osteoblasts. Cells were obtained from fetal rat calvaria by sequential collagenase digestion, and were seeded in 6-well plates at 106 cells/ml in BGJb medium + 10% FCS. The media was then replaced with DMEM serum free and incubated for additional 24 hrs. Cells were then incubated with [3H]oleic acid (5 uCi/m1 for 24 hrs. EGF (50 nM), phorbol 12-myristate 13-acetate (PMA, 1 uM) or alpha-thrombin (8 u/ML) were added to cells in the presence or absence of 5% ethanol. PLD activity was measured by the formation of [3H]phosphatidylethanol (PEtOH). The lipids were extracted and separated by thin layer chromatography (t.l.c). These were then viewed by iodine staining and autoradiography. Samples were then scraped off the t.l.c. plates and transferred to vials for scintillation counting. ANOVA was used for statistical analyses. EGF significantly (p<0.05) stimulated PLD activity at all time periods of incubation studied (20, 45, and 60 secs), but the highest stimulation occurred as at 45 secs of incubation with mitogen, PMA after 3 minutes incubation, which has previously been shown to stimulate protein kinase C (PKC) activity in this cell system, significantly stimulated PLD activity at a greater level than that found for EGF. Thrombin after 60 seconds also significantly stimulated the release of [3H]PEtOH. These results strongly suggest that one mechanism of action of EGF and alpha-thrombin in normal rat osteoblastic cells is the hydrolysis of membrane phosphatidylcholine by the activation of a specific PLD, and that PKC may participate in the regulation of this activity. Key Words: Inositol trisphosphate, Phospholipase-D, Epidermal growth factor, phosyphatidylcholine, Protein Kinase C.